The proposed research is a continued study of group II intron mobility mechanisms and the development of group II introns as vectors for targeted gene disruption and site-specific DNA insertion. Mobile group II introns are catalytic RNAs ("ribozymes") that encode reverse transcriptases and are thought to be evolutionary ancestors of nuclear premRNA introns, non-LTR retrotransposons and telomerase. These introns use a novel mobility mechanism mediated by an RNP particle that is formed during RNA splicing and contains the intron-encoded reverse transcriptase and the excised intron lariat RNA. For mobility, this RNP recognizes a relatively long DNA target site (30-35 bp), with both the protein and base pairing of the intron RNA used for DNA target site recognition. The intron RNA then inserts directly into one DNA strand by reverse splicing, while the intron-encoded protein cleaves the opposite strand and uses the 3' end at the cleavage site as a primer for reverse transcription of the inserted intron RNA. Because most of the DNA target site is recognized by base pairing, group II introns can be retargeted to insert into any desired DNA target simply by modifying the intron RNA. During the current grant period, we used this feature to develop group II introns into highly efficient, controllable gene targeting vectors for both Gram positive and Gram-negative bacteria, including commercially and medically important species that lack good genetic systems. Mobile group II introns are of continued interest because they use a novel RNP-based mechanism for site-specific DNA cleavage and insertion and because they are related evolutionarily to introns, retrotransposable elements, and telomerase in the human genome. Further, as highly efficient bacterial gene targeting vectors, we anticipate that group II introns will have applications in the genetic engineering and functional genomics of a wide variety of bacteria, including the identification of novel drug targets and probiotics, which may lead directly to new therapies for bacterial diseases. Moreover, if group II introns can be adapted to function as efficiently for gene targeting in eukaryotes as has been possible in prokaryotes, they would have widespread applications in functional genomics and gene therapy in higher organisms, again including direct application to the cure of human diseases. Specific aims are: (1) To continue to study group II intron DNA target site recognition, focusing on defining and modifying DNA-protein interactions. (2) To study other key aspects of the group II intron mobility mechanism, including the possibility that the RNPs use facilitated diffusion for DNA target site recognition, how the IEP and intron RNA contribute to DNA unwinding, RNP conformational changes during different steps of mobility, and the identity of the enzymes used for second-strand DNA synthesis. (3) To continue to develop bacterial group II intron gene targeting methods and apply them to medically important bacteria. (4) To develop group I1 intronbased gene targeting methods for eukaryotes, including human cells.